Morphine-6-glucuronide synthesis

ABSTRACT

The invention provides a novel method for synthesising M6G, and intermediates therefor. In order to synthesise M6G the major problem to overcome is to obtain the glycoside linkage with very high β-selectivity since prior methods produce the α-anomer. The invention provides a method for the preferential synthesis of the β-anomer of M6G which includes the step shown in Scheme 6 wherein use of DMAP is optional.

[0001] The invention provides a novel method for synthesisingMorphine-6-Glucuronide (M6G) and intermediates therefor.

[0002] Synthesis of M6G from 3-acetyl-morphineand-methyl2-α-bromo-3,4,5-tri-O-acetylgucuronate is described by Lacy,C., et al. (Tetrahedron Letters, 36 (22), (1995), 3949-3950).

[0003] Hidetoshi, Y. er a!., (Chemical and Pharmaceutical Bulletin, JP,TOKYO, 16 (11), (1968), 2114-2119) describe synthesis of M6G by reactionof 3-acetyl-morphine with a bromo derivative of glucuronic acid to forma Methyl [3-acetyl-morphine-6-y1-2,3,4-tri-O-acetyl-β-D-glucopyranosid]uronate intermediate which is subsequently hydrolysed to M6G.

[0004] WO 93/05057 discloses preparation of M6G by reaction of 3-acetylmorphine with methyl 1α-bromo, 1-deoxy, 2,3,4tri-O-acetyl Dglucopyranuronate and subsequently hydrolysing the resultingintermediate to M6G.

[0005] In order to synthesise M6G the major problem to overcome is toobtain the glycoside linkage with very high β-selectivity since priormethods produce the α-anomer.

[0006] One method for obtaining high β-selectivity is to usetrichloroimidate as the leaving group, as shown in WO 93/03051: FIG. 1(Salford Ultrafine Chemicals and Research Limited).

[0007] Orthoesters are simple to synthesise from their respectivebromides¹. There is a reaction reported in the literature² between theglucuronate orthoester (2) and the sugar derivative (3) catalysed bylutidinium perchlorate³ (4) (Scheme 1).

[0008] When this reaction was repeated with the t-butyl orthoacetate (5)and cyclohexanol (6 equivalents), the desired product (6) was isolatedin 9% yield. Two other products also suggested that they were thedesired product, but with the loss of one acetyl group, isolated in acombined yield of 43% (Scheme 2).

[0009] When 1.2 equivalents of 4-tert-butylcyclohexanol was used, thedesired compound (7) was obtained in 17% yield. Other compounds obtainedfrom the reaction also appeared to contain the desired peaks in the nmr,but after further examination proved to be the product oftransorthoesterification (8) (Scheme 3).

[0010] Reaction of orthoester (5) with protected morphine Initially, 1.2equivalents of 3-TBS protected morphine and the orthoester (5) weredissolved in chlorobenzene and half of the solvent was distilled offbefore 0.1 equivalents of lutidinium perchlorate (4) in chlorobenzenewas added. The solvent was continuously distilled off while freshsolvent was added, and after 2.5 h another compound was formed withsimilar tic properties to the protected morphine. Workup andchromatography gave a compound which corresponded totrans-orthoesterified material (9). None of the desired material wasobtained (Scheme 4).

[0011] This product (9) was resubmitted to the reaction conditions (0.1equivalents of lutidinium perchlorate and protected morphine inrefluxing chlorobenzene) with no new products formed after 4h. Twofurther reactions were attempted using two equivalents of orthoester (5)and 0.2 equivalents of lutidinium perchlorate and 1 equivalent oforthoester (5) and 1.2 equivalents of lutidinium perchlorate, but bothgave varying yields of orthoester (9).

[0012] We have concluded that a different, more bulky, alkyl group wasneeded on the orthoester to hinder attack there. Initially, theisopropyl group was examined. However, the initial reaction,perisobutyrylation, failed to give a compound which recrystallised frompetrol, so the α and β anomers could not be separated. Therefore,attention focussed on the pivaloyl group.

[0013] The invention is further described with reference to theaccompanying FIG. 2 which shows a summary of a reaction scheme accordingto the invention for synthesising M6G

[0014] Synthesis of the Perpivalated Glucuronide

[0015] Synthesis of perpivalated glucuronide proved troublesome atfirst, giving a mixture of 3 and 4 non-pivalated material (scheme 5).

[0016] A search through the literature revealed that glucose can beperpivalated by heating the reaction to reflux for 3h. and then stirringit for 7 days.

[0017] When this-reaction was repeated on ring-openedglucurono-3,6-lactone (Scheme 6), perpivalated product (10)was obtainedby crystallisation of the crude product from MeOH (or EtOH) and waterand drying the crystals by dissolving them in DCM, separating any waterpresent, drying; and then evaporating the organic layer to give theproduct in 29-52% yield, a substantial improvement on previous yieldsfor this step.

[0018] DMAP was added to aid perpivalation, although there has been noevidence to suggest that this is necessary The variation in the yieldsquoted is probably due to the amount of MeOH left over from the firststep The high yield quoted (52%) was obtained by using 6 (instead of 5)equivalents of tBuCOCl. A slight colouration of the final product provedno handicap in the next step, as after a silica plug andrecrystallisation, pure white crystals were obtained. Synthesis of theOrthoester (6)

[0019] Conversion of the perpivalated material (10) to the α-bromide(11) required gentle heating (to approxiamately 35° C.) to dissolve thesubstrate in the reaction mixture. The reaction proceeded very cleanlyby tic analysis, showing 2 spot to spot conversion. Attempts to reducethe amount of HBr used to five equivalents led to incomplete conversionof the starting material, so 12 equivalents were used as before. Theproduct was slowly crystallised from EtOH/water or MeOH/water to givelong white crystals in a yield of 52-78%. High yields were alwaysobtained when fresh HBr/AcOH was used. The crystals were dried by againdissolving them in dichloromethane, the water separated, and the organiclayer dried and evaporated.

[0020] The orthoester(12) was obtained in 63-81% yield by stirring a 1:1mixture of EtOH:collidine at 70° C. (oil bath temperature) with thebromide (11) and 0.8 equivalents of Et₄NBr (Scheme 7). The product caneasily be crystallised from EtOH/water water or MeOH/water as whitecrystals, with a trace of collidine still present (detected by smell!)but which doesn't effect the next reaction. An interesting by-productfrom this reaction (obtained in about 10%) is the result of EtOHattacking the anomeric position to give the β-anomer (13) Again, thedifficulty in drying the crystals meant that they were dissolved inpetrol (40-60), the water separated, and the organic layer dried andevaporated.

[0021] Synthesis of 3-Pivalated morphine (14)

[0022] Selective deprotonation of the phenolic OH of morphine wasachieved using NaH (surprisingly, the anion turns out to be soluble inTHF) and trimethylacetyl chloride was added dropwise to give the desiredproduct after recrystallisation from MeOH/water (Scheme 8). Again, thedifficulty in drying the crystals meant that they were dissolved indichloromethane, the water separated, and the organic layer dried andevaporated to give a white powder in 81% yield.

[0023] 1.1 equivalents of trimethylacetyl chloride were used, but thisled to some dipivalated morphine which proved difficult, torecrystallise apart from mono-pivalated morphine (14) or the protectedM6G (16). Thus, it would be advantagous in the future to use 1equivalent of trimethylacetyl chloride.

[0024] Synthesis of Lutidinium Perchlorate (15)

[0025] This was achieved by simply adding aqueous perchloric acid to anether solution of lutidine (excess, as this remains in the Et2O layer)(Scheme 9 ) and evaporating the water until crystals form, which werecollected by filtration.

[0026] The crystals are deliquescent and thus need to be dried underhigh vacuum prior to use.

[0027] Other acid catalysts have been investigated in the couplingreaction below, but with no success. However, this compound has shown notendencies to decompose, proving both thermal and shock stable, soshouldn't prove a problem on scale up.

[0028] Coupling of the orthoester (12)with 3-pivalated morphine (14)

[0029] Coupling the orthoester (12) to 1.1 equivalents of 3-pivalatedmorphine (14) was achieved by adding 0.1 equivalents of lutidiniumperchlorate (15)every 15 min. until 1.2equivalents had been added to thedistilling chlorobenzene. The reaction was then stirred under reflux fora further 2h. to give a mixture of 3-pivalated morphine (14) protectedM6G (16) and much less polar materials. Work-up and crude purificationby chromatography gave protected M6G (16) and 3-pivalated morphine (14)which was purified by recrystallisation from MeOH/(water, smallquantity) to give (16) in 29% yield (with no detectable quantity ofα-anomer or trans-orthoesterified material from nmr analysis) (Scheme10).

[0030] This yield is the greatest amount obtained from this reaction andfurther improvements might be possible. Lutidinium perchlorate (15)wasadded every 15 min. as a solid appeared to crystallise from the reactionmixture (presumably the 3-pivalated morphine perchlorate) and, if nomore catalyst is added, the major product turned out to be thetrans-orthoesterified material (similar to orthoester (9) in Scheme 14).If 1.2 equivalents of lutidinium perchlorate (15)was added directly tothe reaction, only 6% of coupled material was obtained (presumably asall the 3-pivalated morphine had been removed from the reaction as theperchlorate salt). The main problem with adding the catalyst, is itsinsolubility in chlorobenzene lower than approxiamately 100° C. If it ispossible on a large scale to add lutidinium perchlorate(15) inchlorobenzene a; 1000° C., this may prove not only simpler to add thecatalyst, but also lead to increasing yields. The reaction also needs tobe refluxed for an additional 2 hours after all the lutidiniumperchlorate (15) has been added, to cause the trans-orthoesterifiedmaterial to rearrange to the desired material.

[0031] Global Deprotection of Protected M6G (16)

[0032] Heating protected M6G (16) in MeOH until it dissolves beforeadding the water (which causes it to crystallise from the reactionmixture) and Ca(OH)₂ seems to be the mildest way of performing thisreaction. After stirring for 3 days, the reaction gave, by tic analysis,M6G (17) and morphine (Scheme 11).

[0033] The reaction was quite slow due to the insolubility of Ca(OH)₂ inwater, but when the reaction is deemed to have finished by tic analysis,6.5 equivalents of sulfuric acid were added or until the reactionreached pH 4. The CaSO₄ so formed was filtered off and thetrimethylacetic acid also formed was removed by washing the filtratewith DCM. Evaporating the water proved the hardest part of this reactiondue to excessive foaming. Some CaSO₄ remains in the filtrate and thiswas removed by adding MeOH to crystallise it out. The residue producedafter all the water had been evaporated was purified by repeated washingwith MeOH as M6G is virtually insoluble in MeOH while morphine issoluble in it. The morphine present in the crude residue probablyarrived there due to the di-pivalated morphine passing through thecoupling reaction and then being deprotected to morphine in this finalstep Hopefully, by using strictly 1 equivalent or trimethylacetylchloride, this should eliminate the di-pivalated morphine, thus makepurification of M6G even simpler, and increasing the yield for the finalstep

[0034] The invention is further described in detail below by way ofexample only.

EXAMPLE 1

[0035] Methyl 1β,2,3,4-tetra-O-pivaloylglucuronate

[0036] Glucurono-6,3-lactone (147 g, 0.8 mol) was stirred as asuspension in methanol (1 L, not dried) under nitrogen. A catalyticamount of sodium methoxide (147 mg, 2.6 mmol) was added to thesuspension, and after 2 hours most of the suspension was still present.The reaction proceeded very slowly at room temperature, ˜18° C., butnoticeably increased in rate when the reaction was warmed, therefore,the reaction was gently warmed to ˜25° C. After another hour ofstirring, most of the suspension had dissolved to leave a clear yellowsolution that was then evaporated. The residue was found to be a solid,which tended to foam under vacuum, which made total removal of all themethanol difficult.

[0037] Chloroform (400 mL), followed by 6 equivalents of pyridine (400mL, 4.8 mol) and a catalytic amount of N,N-dimethylg-4aminopyridine (4g) was then added to the residue that slowly dissolved in this mixtureThe solution was stirred using a magnetic stirrer plate and flea, butthe problems encountered in continuously stirring this reaction wouldmake an overhead mechanical stirrer preferable at this stage. Thereaction was then cooled to 0° C. and 5 equivalents of trimethylacetylchloride (500 mL, 4 mol) was added gradually, not allowing the reactionto warm to a temperature above ˜8° C. The yellow/orange solution becamecolourless on addition of the first portion of trimethylacetyl chloride,and alter approximately half of the volume was added, a whiteprecipitate was observed (pyr.HCl) After addition was complete, thereaction was stirred overnight at room temperature before being heatedat reflux for 2 hours, during which time the reaction turned black withthe white precipitate still present. Tlc analysis showed that thedesired product had been produced (Rf 0.5, 1:1 Et₂O:petrol), but somemono-unprotected material remained (Pf 0.3 and 2.8, 1.1 Et₂O:petrol).The reaction was then allowed to cool to room temperature over 3 hours,then further cooled to 0° C. before methanol was added gradually (thisquenches the excess trimethylacetyl chloride to give methyltrimethylacetate, which is evaporated off with the solvent). The blacksolution was then poured into a 2L separating funnel, and washed withwater (600 mL), 1M HCl (2 * 600 mL), water (600 mL), and saturatedaqueous NaHCO₃ (2 * 600 mL). The organic layer was then dried with MgSO₄and passed through approximately 5cm of silica on a sinter funnel (whichremoved a black baseline compound). The silica was washed withdichloromethane (100 mL) and the combined filtrates evaporated to leavea black viscous oil, which was re-dissolved in ethanol (˜1 L) and hadwater added until the solution turned turbid (˜500 mL). More ethanol wasadded until the turbid solution cleared, and the solution was left tocrystallise overnight. The yellow crystals were dissolved indichloromethane (300 mL) and any excess water removed by separation, thedichloromethane layer Was then dried and evaporated.

[0038] The white powder (113.5 g, 26%) was then used in the nextreaction.

[0039] Methyl 1-deoxy-1-α-bromo,2,3,4-tri-O-pivaloylglucuronate

[0040] Methyl 1(β),2,3,4-tetra-O-pivaloylglucuronate (108.5 g, 0.2 mol)was dissolved in glacial acetic acid (500 mL) (with the aid of somegentle heating) and placed in a bath of cold water. 12 equivalents of33% HBr in acetic acid (500 mL, 2.9 mol) were then added at a raterequired to prevent the acetic acid freezing without the reactionexotherning too greatly. After the addition was complete, the reactionwas allowed to warm to room temperature. If any white solid (startingmaterial) persisted, gentle warming was applied to the reaction until itdissolved and the reaction then allowed to cool and stir overnight. Theorange/brown solution was then cautiously poured into dichloromethane(500 mL)/water (500 mL), the organic layer separated, washed with water(500 mL) and saturated NaHCO3 (500 mL) (with care to avoid too rapid anevolution of C0₂). The organic layer was then dried (MgSO₄) and passedthrough approximately 2 cm of silica, the silica was washed with moredichloromethane (50 mL) and the combined filtrates evaporated (takingcare to remove all the dichloromethane). The residue was then dissolvedin EtOH (˜400 mL) and water added until the reaction turned turbid. Moreethanol was added until the solution just turned clear and the productallowed to crystallise overnight which were collected by filtration. Thecrystals were dissolved in dichloromethane and the organic layerseparated from any water that remained, dried (MgSO₄), and evaporated

[0041] The white powder (76 g, 72%) was then used in the next reaction.

[0042] Methyl 1β,2-ethylordhopivalate-3,4-di-O-pivaloylglucuronate

[0043] Methyl 1-deoxy-1-α-bromo,2,3,4-tri-O-pivaloylglucuronate (69 g,0.13 mol) was dissolved in collidine (300 mL) (pre-dried by distillingonto activated 3 Å sieves) and ethanol (300 mL) (pre-dried by distillingfrom NaOEt onto activated 3 Å sieves). 0.8 equivalents of pre-driedtetraethylammonium bromide (22 9, 0.1 mol) was then added to thereaction, which was stirred at 60° C. (oil bath temperature 70° C.)overnight. The reaction was then cooled and poured into dichloromethane(500 mL)/water (500 mL) and the organic layer separated, dried (MgSO₄),and evaporated. The collidine was removed by low-pressure distillation(total evaporation is not necessary), the residue dissolved in EtOH(˜400 mL), and water added until the product started to crystallise out.The white crystals were collected by filtration and dissolved in petrol.The organic layer was then separated from any water that remained, dried(MgS0₄), and evaporated.

[0044] The white powder (50 g, 78%) was then used in the next reaction

[0045] 3-O-Pivaloylmorphine

[0046] Morphine (12g, 42 mmol) was added portionwise to a THF (80 mL, Nadried) suspension of 1.05 equivalents of petrol washed NaH (60%dispersion in oil, 1.768 g, 44 mmol) a; 0° C. After stirring for 1 h atroom temperature, 1.1 equivalents of trimethylacetyl chloride (5.7 mL,46 mmol) were added to the clear reaction mixture at 0° C. andeventually a white solid precipitated from the reaction. After 1 h.,MeOH (10 mL) followed by saturated aqueous sodium bicarbonate (100 mL)were added to the reaction which was then extracted with Et₂O (2×200mL). The combined extracts were washed with brine (200 mL), dried, andevaporated. The residue was recrystallised from MeOH/water and thecrystals dissolved in dichloromethane and the organic layer separatedfrom any water that remained, dried (MgSO₄), and evaporated.

[0047] The white powder (12.6 g, 81%) was then used in the nextreaction.

[0048] Lutidinium Perchlorate

[0049] A 60% aqueous solution of perchloric acid (29 mL, 0.27 mol) wasadded to 1.1 equivalents of lutidine (34 mL, 0.29 mL) in Et₂O (250 mL)at 0° C. After stirring for 0.5 h. at room temperature, the aqueouslayer was separated and the water evaporated until a white solidcrystallised from the water, the crystals filtered off and washed withEt₂O to give the product as a white crystalline solid (30 9, 54%). Theproduct was dried under high vacuum prior to use.

[0050] Methyl1β-6′-O-(3′-O-pivaloylmorphine)-2,3,4-tri-O-pivaloylglucuronate

[0051] A chlorobenzene (400 mL) (distilled from CaH₂ onto activated 3Åsieves) solution of 1.1 equivalents of 3-O-pivaloyoloxymorphine (8.49 g,23 mmol) and methyl 1α,2-ethylorthopivalate-3,4di-O-pivaloylglucuronate(10 g, 20 mmol) was heated to reflux to distil off approximately half ofthe solvent. 0.1 Equivalents of lutidinium perchlorate (415 mg, 2 mmol)was then added to the reaction that was still at reflux. The reactionwas then stirred at reflux for 15 min with chlorobenzene continuouslydistilled off and fresh chlorobenzene added. After this time, a further0.1 equivalents of lutidinium perchlorate (415 mg, 2 mmol) was thenadded to the reaction. This procedure was repeated every 15 min until1.2 equivalents of lutidinium perchlorate (5.2 g, 25 mmol) had beenadded. The reaction was then stirred at reflux for 2 hours withchlorobenzene continuously distilled off and fresh chlorobenzene added.After this time, the reaction was allowed to cool and then poured intodichloromethane (500 mL)/water (500 mL), the organic layer separated,washed with saturated aqueous sodium bicarbonate (500 mL), dried, andevaporated. The residue, after some of the chlorobenzene had beenremoved under low pressure, was applied to the top of a silica columnand eluted with diethyl ether to remove the non-polar by-products andthen with 5% methanol in dichloromethane. The desired product wasseparated from 3-Piv-M by recrystallisation from MeOH/water to give awhite crystalline powder (4.76 g, 29%)

[0052] Morphine-6-gluconoride

[0053] Methyl1β-6′-O-(3′-O-pivalayloxymorphine)-2,3,4-tri-0-pivaloylglucuronate (3.06g, 3.77 mmol) was dissolved in MeOH (60 mL) (with the help of someheating) and had water (7 mL) followed by 6.5 equivalents of calciumhydroxide (1.817 g, 24.5 mmol) added to it. The reaction was stirred fortwo days when water (60 mL) was added and the reaction stirred for afurther day until the reaction was shown to be complete by tic analysis(Rf 0.3, 45% nBuOH; 15% water; 20% acetone; 10% acetic acid; 10% of a 5%aqueous solution of arnmonia). 6.5 equivalents of 0.25 M aqueoussulphuric acid (98 mL, 24.5 mmol) were added (pH 4) and the reactionstirred for 1 hour. The reaction was then filtered to remove CaSO₄ andthe solid washed with water (30 mL). The filtrate was then washed withDCM (2×100 mL), three quarters of the water evaporated and the samequantity of MeOH added. The white solid (mainly CaSO₄) was then filteredand the filtrate evaporated. The residue (1.56 g) had MeOH (100 mL)added and the white solid filtered and repeatedly washed with MeOH togive the desired compound (1.05 g, 60%) which could, according to theliterature, be recrystallised from H₂O/MeOH (although this has not beenperformed on this material).

[0054] References

[0055] 1. For a review of orthoesters and their synthetic applicationssee N. K. Kochetkav and A. F. Bochkov, Recent Developments in theChemistry of Natural Carbon Compounds, Ed. R. Bognar, V. Bruckner, andCs. Szantay, Akademiai Kiado: Budapest, 1971, vol. 4, p.77 191.

[0056] 2. H. P. Wessel, L. Labler, and T. S. Tschopp, Helv. Chim. Acta.,1989, 72, 1268.

[0057] 3. The use of 2,6-dimethylpyridinium perchlorate (4) was firstreported by N. K. Kochetkov, A. F. Bochkov, T. A. Sokolovskaya, and V.J. Snyatkova, Carbohydr. Res.,1971,16, 17.

1. A method for the preferential synthesis of the β-anomer of M6G whichincludes the step shown in Scheme 10:


2. Synthesis according to claim 1 which includes the step shown inScheme 7:


3. Synthesis according to claim 1 or 2 which includes the step shown inScheme 6:

wherein use of DMAP is optional,
 4. Synthesis according to any precedingclaim which includes the step shown in scheme 8:


5. Synthesis according to any preceding claim which includes the stepshown in Scheme 9:


6. Synthesis according to any preceding claim which includes a step tohydrolyse the protecting groups from compound
 16. 7. Synthesis accordingto claim 6 in which the hydrolysis is as shown in Scheme 11:


8. A compound of formula (12) as defined in claim 2 or a derivativethereof for use in a method according to claim
 1. 9. Use of a compoundof formula (10) as defined in claim 1 or a derivative thereof in amethod according to claim
 2. 10. Use of a compound of formula (11) asdefined in claim 2 or a derivative thereof in a method according toclaim
 2. 11. Use of a compound of formula (14) as defined in claim 3 ora derivative thereof in a method according to claim
 1. 12. Use of acompound of formula (15) as defined in claim 4 or a derivative thereofin a method according to claim
 1. 13. Use of a compound of formula (16)as defined in claim 5 or a derivative thereof in a method according toclaim 7.